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Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Dixon SD, Huynh MM, Tamilselvam B, Spiegelman LM, Son SB, Eshraghi A, Blanke SR, Bradley KA - PLoS ONE (2015)

Bottom Line: In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type.Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response.In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

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CdtC Mediates Cholesterol Dependency of Ec-CDT.CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH2AX by laser scanning cytometry as in Fig 2B. Data were normalized against pH2AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.
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pone.0143977.g003: CdtC Mediates Cholesterol Dependency of Ec-CDT.CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH2AX by laser scanning cytometry as in Fig 2B. Data were normalized against pH2AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.

Mentions: To determine whether host cellular cholesterol influences intoxication by Ec-CDT, we challenged CHO-A745 cells with holotoxin or CdtAB in the presence or absence of the cholesterol-extracting agent methyl-β-cylcodextrin (mβCD). Intoxication was determined by measuring pH2AX in order to avoid complications associated with reduced cell viability induced by continued presence of mβCD over the longer time course required for cytotoxicity assays (not shown). Cholesterol extraction resulted in a loss of intoxication by Ec-CDT holotoxin (Fig 3), consistent with previous reports for Hd-CDT, Aa-CDT, and Cj-CDT [17,38,39,41]. Interestingly, intoxication by Ec-CdtAB was similarly sensitive to mβCD, suggesting that Ec-CdtA dictates, at least in part, a role for cholesterol in intoxication by Ec-CDT.


Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Dixon SD, Huynh MM, Tamilselvam B, Spiegelman LM, Son SB, Eshraghi A, Blanke SR, Bradley KA - PLoS ONE (2015)

CdtC Mediates Cholesterol Dependency of Ec-CDT.CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH2AX by laser scanning cytometry as in Fig 2B. Data were normalized against pH2AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.
© Copyright Policy
Related In: Results  -  Collection

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pone.0143977.g003: CdtC Mediates Cholesterol Dependency of Ec-CDT.CHO-A745 cells were seeded at 8 x 103 cells/well on 96-well plates and allowed to adhere overnight. The next day, cells were incubated with or without 5 mM MβCD and/or 12.5 μM EGA for 1 h then challenged with 1 μM Ec-CDT or Ec-CdtAB for 16 h. Intoxication was assessed by measuring pH2AX by laser scanning cytometry as in Fig 2B. Data were normalized against pH2AX signal induced by Ec-CDT holotoxin (maximum signal) in each experiment. Graphs represent average values and SEM from three independent experiments, each performed in triplicate. All statistical analyses are from the pairwise post-test (Tukey’s) derived from one-way ANOVA. (Prism 5, GraphPad). Symbols above each column reflect comparison to Ec-CDT holotoxin (ns = not significant; * p < 0.001). Additional pairwise comparisons are indicated by brackets.
Mentions: To determine whether host cellular cholesterol influences intoxication by Ec-CDT, we challenged CHO-A745 cells with holotoxin or CdtAB in the presence or absence of the cholesterol-extracting agent methyl-β-cylcodextrin (mβCD). Intoxication was determined by measuring pH2AX in order to avoid complications associated with reduced cell viability induced by continued presence of mβCD over the longer time course required for cytotoxicity assays (not shown). Cholesterol extraction resulted in a loss of intoxication by Ec-CDT holotoxin (Fig 3), consistent with previous reports for Hd-CDT, Aa-CDT, and Cj-CDT [17,38,39,41]. Interestingly, intoxication by Ec-CdtAB was similarly sensitive to mβCD, suggesting that Ec-CdtA dictates, at least in part, a role for cholesterol in intoxication by Ec-CDT.

Bottom Line: In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type.Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response.In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Show MeSH
Related in: MedlinePlus